Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

To isolate neutrophil subpopulations, including cytotoxic high-density neutrophils, HDNs, and low-density neutrophils, LDNs, from the blood of a tumor-bearing mouse, take a tube containing the mouse blood.

Dilute with buffer containing bovine serum albumin, BSA, proteins, which maintain the osmotic balance and stabilize the blood cells. Layer an appropriately diluted blood volume on a discontinuous sucrose gradient comprising two non-overlapping sucrose layers with differing densities. 

During centrifugation, the cells migrate to different regions in the gradient based on their densities relative to the gradient medium. The majority of the high-density erythrocytes sediment to the tube bottom.

HDNs with granulocytes form a white-red ring at the interface between the sucrose layers, while the LDNs and mononuclear cells form a white ring at the interface between the low-density sucrose layer and the BSA-containing buffer layer.

Remove the top BSA-containing buffer layer. Transfer the low-density cell layer to a tube with BSA-containing buffer. Remove the low-density sucrose layer. Transfer the high-density cell layer to a tube with BSA-containing buffer.

Centrifuge the tubes. Resuspend the cell pellets in sterile water to lyse the erythrocytes. Add a buffer with a higher BSA concentration to restore the cell isotonicity. Centrifuge the tubes. Discard the supernatant containing the lysed erythrocytes.

Resuspend the cells containing LDNs and HDNs in BSA-containing buffer for further analysis.